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circDlgap4 Alleviates Cerebral Ischaemic Injury by Binding to AUF1 to Suppress Oxidative Stress and Neuroinflammation

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Abstract

Ischaemic stroke is one of the most common causes of mortality and morbidity.circDlgap4 has been implicated in ischemia/reperfusion injury through an unknown mechanism. Here, we studied the function of circDlgap4/AUF1 in ischaemic stroke and its underlying molecular mechanism. N2a cells and primary mouse cortical neurons were subjected to OGD to mimic neuronal injury during ischemia. BV-2 cells were treated with LPS to mimic neuroinflammation. The MTT assay was used to assess cell viability, while flow cytometry was used to measure cell apoptosis. qRT–PCR, western blotting, immunohistochemistry, and immunostaining were employed to determine the levels of circDlgap4, AUF1, NRF2/HO-1, proinflammatory cytokines, NF-κB pathway–related proteins, and IBA-1. RIP and RNA pulldown assays were employed to validate the interactions of circDlgap4/AUF1, AUF1/NRF2, and AUF1/cytokine mRNAs. mRNA degradation was used to determine the effects on mRNA stability. The tMCAO model was used as an in vivo model of ischaemic stroke. TCC staining and neurological scoring were performed to evaluate ischaemic injury. circDlgap4 was decreased following OGD and during tMCAO. circDlgap4 overexpression inhibited OGD-induced cell death and oxidative stress and LPS-induced increases in proinflammatory cytokines by increasing NRF2/HO-1. Knockdown of AUF1 blocked the effects of circDlgap4 overexpression. Mechanistically, RIP, RNA pulldown, and mRNA degradation assay results showed circDlgap4/AUF1/NRF2 mRNA formed a complex to stabilize NRF2 mRNA. Furthermore, AUF1 directly interacted with TNF-α, IL-1β, and COX-2 mRNAs, and circDlgap4/AUF1 binding promoted the degradation of these mRNAs. Finally, circDlgap4 ameliorated ischaemic injury in vivo. circDlgap4 alleviates ischaemic stroke injury by suppressing oxidative stress and neuroinflammation by binding to AUF1.

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Data Availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

circRNAs:

Circular RNAs

AUF1:

AU-rich element RNA-binding protein 1

DMEM:

Dulbecco’s modified Eagle medium

OGD:

Oxygen and glucose deprivation

LPS:

Lipopolysaccharide

PI:

Propidium iodide

ELISA:

Enzyme-linked immunosorbent assay

RIP:

RNA Immunoprecipitation

tMCAO :

Transient middle cerebral artery occlusion

TTC:

Triphenyltetrazolium chloride

ROS:

Reactive oxygen species

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Acknowledgements

Thanks to the members of our laboratory for their contributions.

Funding

This work was supported by the National Natural Science Foundation of China (81671296).

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Authors and Affiliations

  1. Department of Neurology, Xiangya Third Hospital, Central South University, No. 138 tongzipo Road, Yuelu District, Changsha, 410013, Hunan Province, China

    Huan Liu, Wen Zheng & Zhi Song

Authors
  1. Huan Liu
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  2. Wen Zheng
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  3. Zhi Song
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Contributions

HL: methodology, data curation, visualization, investigation, validation, writing—original draft preparation; WZ: methodology, software, investigation; ZS: supervision, conceptualization, writing—reviewing and editing.

Corresponding author

Correspondence to Zhi Song.

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All animal experiments and protocol have been approved by the Animal Care and Use Committee of the Third Xiangya Hospital of Central South University.

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The informed consent obtained from study participants.

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The authors declare no competing interests.

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Liu, H., Zheng, W. & Song, Z. circDlgap4 Alleviates Cerebral Ischaemic Injury by Binding to AUF1 to Suppress Oxidative Stress and Neuroinflammation. Mol Neurobiol 59, 3218–3232 (2022). https://doi.org/10.1007/s12035-022-02796-5

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  • DOI: https://doi.org/10.1007/s12035-022-02796-5

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